Albumin-free botulinum toxin formulations

ABSTRACT

This invention relates to botulinum toxin formulations that are stabilized without the use of any proteinaceous excipients. The invention also relates to methods of preparing and using such botulinum toxin formulations.

This application is a divisional of patent application U.S. Ser. No.12/824,118, filed on Jun. 25, 2010, now U.S. Pat. No. 9,340,587, issuedon May 17, 2016, which claims benefit of U.S. Provisional PatentApplication No. 61/220,433, filed on Jun. 25, 2009, the contents of allof which are incorporated herewith by reference in their entireties.

FIELD OF THE INVENTION

This invention relates to pharmaceutical formulations containingbotulinum toxin. In particular, this invention relates to botulinumtoxin formulations that are stabilized by non-proteinaceous excipients.

BACKGROUND OF THE INVENTION

Botulinum toxins (also known as botulin toxins or botulinum neurotoxins)are neurotoxins produced by Clostridium botulinum bacteria. Botulinumtoxins produce paralysis of muscles by preventing synaptic transmissionor release of acetylcholine across the neuromuscular junction. Theaction of botulinum toxins essentially blocks signals that normallywould cause muscle spasms or contractions, resulting in paralysis.

There are eight naturally occurring serologically related botulinumtoxins, seven of which are known to cause paralysis (viz., botulinumneurotoxin serotypes A, B, C, D, E, F and G). Each of these serotypes isdistinguished by neutralization with type-specific antibodies. However,the molecular weight of the botulinum toxin protein molecule is about150 kD for all seven of these active botulinum toxins. As released bythe Clostridium botulinum bacteria, the botulinum toxin is present in acomplex comprising the 150 kD botulinum toxin protein molecule alongwith associated non-toxin proteins. The total size of the complex mayvary. For instance, the botulinum toxin type A complex can be producedby Clostridium botulinum bacteria as 900 kD, 500 kD and 300 kDcomplexes. Botulinum toxin types B and C complexes are only produced as700 kD or 500 kD complexes. Botulinum toxin type D complexes areproduced as both 300 kD and 500 kD complexes. Botulinum toxin types Eand F complexes are only produced as 300 kD complexes. The complexes arebelieved to contain non-toxin hemagglutinin protein and non-toxin andnon-toxic non-hemagglutinin protein. These two non-toxin proteins (whichalong with the botulinum toxin molecule comprise the relevant neurotoxincomplex) are believed to provide stability against denaturation to thebotulinum toxin molecule and protection against digestive acids whentoxin is ingested.

While botulinum toxin is the most lethal naturally occurring toxin knownto man, it has found extensive use as both a therapeutic and a cosmeticagent. For example, in 1986, the feasibility of using type A botulinumtoxin for treatment of movement-associated wrinkles in the glabella areawas first demonstrated by Schantz and Scott, in Lewis G E (Ed)Biomedical Aspects of Botulinum, N.Y.: Academic Press, 143-150 (1981).The use of botulinum type A for the treatment of wrinkles was publishedin 1992 (Schantz and Scott, in Lewis G. E. (Ed) Biomedical Aspects ofBotulinum, N.Y.: Academic Press, 143-150 (1981)), and by 1994, othermovement-associated wrinkles on the face were being treated with type Abotulinum toxin (Scott, Ophthalmol, 87:1044-1049 (1980)). The demand forcosmetic botulinum toxin treatments has grown steadily over the years,with current annual sales of botulinum toxin in the United Statesexceeding $1 billion dollars per year.

One challenging aspect of manufacturing commercial botulinum toxinformulations is stabilizing the botulinum toxin. Like many proteins,botulinum toxin may be degraded or denatured by environmental factors,such as heat, alkaline conditions, mechanical shear forces, or contactwith reactive surfaces or substances. Furthermore, the difficulty instabilizing the botulinum toxin in commercial formulations isexacerbated by the extreme toxicity of the toxin, which permits onlyminute amounts of toxin to be used for therapeutic purposes. If thebotulinum toxin formulation is not properly stabilized, the minuteamounts of botulinum toxin may undergo unwanted reactions and/or adhereto the inner surfaces of its storage containers, leading to unacceptableloss of botulinum toxin or activity.

Commercial botulinum toxin formulations are often distributed aslyophilized (i.e. freeze dried) or vacuum-dried powder, in order toprevent degradation and make the botulinum toxin formulation easier tohandle and less expensive to transport. Prior to use, botulinum toxinpowder formulations are reconstituted with a liquid carrier, such aswater or a saline solution. For instance, one commercially availablebotulinum toxin formulation is sold under the trademark BOTOX®(Allergan, Inc., Irvine, Calif.). The BOTOX® formulation is distributedas a vacuum-dried powder stored in individual vials, each of whichcontains about 100 units (U) of Clostridium botulinum toxin type Acomplex, 0.5 milligrams of human serum albumin and 0.9 milligrams ofsodium chloride. It has been reported that commercial botulinum toxinformulation must be stored at a temperature of −10° C. or less tomaintain the labeled potency for the one year shelf life.

In commercial formulations of botulinum toxin, human serum albumin isoften added as a bulk carrier and stabilizer. Generally, albumin maystabilize a therapeutic protein (e.g., botulinum toxin) by one or moreof the following mechanisms: (1) reducing adhesion of the therapeuticprotein to the inner surfaces of storage or dispensing containers, whichinclude glassware, storage vials, and the syringe used to inject thepharmaceutical composition; and (2) reducing denaturation of thetherapeutic protein, especially after reconstituting to prepare asolution of the therapeutic protein. Human serum albumin has the addedadvantage of being minimally immunogenic, which lessens the likelihoodthat a human patient will develop antibodies against the botulinum toxinformulation.

Although human serum albumin has been adopted as a stabilizer incommercial botulinum toxin formulations, there are still significantproblems associated with this approach. One serious problem is thatalbumin is derived from blood and is therefore susceptible to carryingblood borne pathogens or infectious agents. For instance, the humanserum albumin may carry the Human Immunodeficiency Virus (HIV). Albuminmay also carry prions, which are proteinaceous infectious agents thatare responsible for causing a neurodegenerative disorder known asCreutzfeldt-Jacob disease. The prions cause misfolding of proteins inthe brain, resulting in dementia, memory loss, speech impairment, lossof motor coordination, and death, often within the span of months afterthe initial onset of symptoms.

Attempts to replace human serum albumin with non-proteinaceousstabilizers generally have been met with difficulties. Anon-proteinaceous polymer may be reactive towards the botulinum toxin ormay contain reactive impurities that degrade and/or denature thebotulinum toxin. For example, some studies have used a poloxamer as anon-proteinaceous stabilizing compound for botulinum toxin. However,these studies report that reconstituted poloxamer-stabilized botulinumtoxin formulations demonstrate low botulinum toxin activity, suggestingthat the poloxamer excipient either failed to properly stabilize thebotulinum toxin and/or induced unwanted degradation reactions to occur.

Accordingly, it would be highly desirable to have a botulinum toxinformulation that is stabilized, but without a proteinaceous excipient,especially without any animal-protein based excipients. Furthermore, itwould be highly desirable to have a non-proteinaceous stabilizingexcipient that does not itself react with botulinum toxin.

SUMMARY OF THE INVENTION

In one aspect, this invention relates to botulinum formulations that arestabilized by non-proteinaceous excipients. In particular, in preferredembodiments, this invention relates to botulinum toxin formulations thatare stabilized without albumin or other animal protein-derivedexcipients.

One aspect of the invention is to provide a liquid compositioncomprising a botulinum toxin, a non-reducing disaccharide or anon-reducing trisaccharide, a non-ionic surfactant, and aphysiologically compatible buffer capable of maintaining the pH between4.5. and 6.5. The concentration of the non-reducing sugar in the liquidcomposition is in the range of 10% through 40% (w/v) and theconcentration of the non-ionic surfactant is in the range of 0.005%through 0.5% (w/v).

Another aspect of the invention is to provide a powder composition bydrying the liquid composition described above.

This invention also provides a method for stabilizing a botulinum toxinformulation. The method comprises combining a botulinum toxin, anon-reducing disaccharide or a non-reducing trisaccharide, a non-ionicsurfactant, and physiologically compatible buffer components capable ofmaintaining the pH between 4.5 and 6.5 to form a liquid composition. Theconcentration of the non-reducing sugar in the liquid composition is inthe range from 10% to 40% (w/v) and the concentration of the non-ionicsurfactant is in the range from 0.005% to 0.5% (w/v). Optionally, themethod further comprises drying the liquid composition to produce astabilized powder composition.

Another aspect of the invention is to provide a liquid compositioncomprising a botulinum toxin, a non-reducing disaccharide or anon-reducing trisaccharide, a non-ionic surfactant, a bulking agent anda physiologically compatible buffer. In these embodiments, theconcentration of the non-reducing disaccharide compositions is in therange of 0.50 to 3.0% (w/v), the concentration of the bulking agent isin the range of 1.5% to 7.5%, the concentration of the non-ionicsurfactant is in the range of 0.005% to 0.5% (w/v); and the pH of thecomposition is in the range of 4.5 to 6.5. Also, the amount ofnon-reducing disaccharide or non-reducing trisaccharide relative to theamount of bulking agent is selected such that the bulking agent does notcrystallize when the liquid composition is dried. The invention alsoexpressly contemplates powder formulations prepared by drying suchliquid compositions.

The invention also provides a method for stabilizing a botulinum toxinformulation comprising combining a botulinum toxin, a non-reducingdisaccharide or a non-reducing trisaccharide, a non-ionic surfactant, abulking agent and a physiologically compatible buffer to form a liquidcomposition. The concentration of the non-reducing disaccharidecompositions is in the range of 0.50 to 3.0% (w/v), the concentration ofthe bulking agent is in the range of 1.5% to 7.5%, the concentration ofthe non-ionic surfactant is in the range of 0.005% to 0.5% (w/v); andthe pH of the composition is in the range of 4.5 to 6.5. Optionally, themethod further comprises drying the liquid composition to produce astabilized powder composition. Also, the amount of non-reducingdisaccharide or non-reducing trisaccharide relative to the amount ofbulking agent is selected such that the bulking agent does notcrystallize when the liquid composition is dried.

This invention also provides a liquid composition comprising a botulinumtoxin, a non-reducing sugar, a non-ionic surfactant, a physiologicallycompatible buffer, a positively charged peptide and an optional bulkingagent. The positively charged peptide has an amino acid sequenceselected from the group consisting of RKKRRQRRR-G-(K)₁₅-G-RKKRRQRRR,RGRDDRRQRRR-G-(K)₁₅-G-RGRDDRRQRRR (SEQ ID NO: 2), andYGRKKRRQRRR-G-(K)₁₅-G-YGRKKRRQRRR(SEQ ID NO: 3). The pH of the liquidcomposition is in the range of 4.5 to 6.5. The invention also expresslycontemplates powder formulations prepared by drying such liquidcompositions.

Yet another aspect of the invention is to provide a method forstabilizing a botulinum toxin formulation comprising combining abotulinum toxin, a non-reducing sugar, a non-ionic surfactant, aphysiologically compatible buffer, a positively charged peptide and anoptional bulking agent. The positively charged peptide has an amino acidsequence selected from the group consisting ofRKKRRQRRR-G-(K)₁₅-G-RKKRRQRRR (SEQ ID NO: 1),RGRDDRRQRRR-G-(K)₁₅-G-RGRDDRRQRRR (SEQ ID NO: 2), andYGRKKRRQRRR-G-(K)₁₅-G-YGRKKRRQRRR (SEQ ID NO: 3). The pH of the liquidcomposition is in the range of 4.5 to 6.5. Optionally, the methodfurther comprises drying the liquid composition to produce a stabilizedpowder composition.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: Recovered botulinum toxin activity as a function of time afterstorage of a lyophilized botulinum toxin at 40° C.

FIG. 2: Recovered botulinum toxin activity as a function of time afterstorage of a lyophilized botulinum toxin at different temperatures.

FIG. 3: Recovered botulinum toxin activity as a function of time afterstorage of a lyophilized botulinum toxin at different temperatures.

DETAILED DESCRIPTION OF THE INVENTION

This invention relates to botulinum toxin formulations that arestabilized without the addition of proteinaceous excipients derived(i.e., purified) from animal sources, such as albumin. As such, thebotulinum formulations of the invention do not suffer from the potentialproblems associated blood borne pathogens or other types of infectiousagents. This invention also provides methods for preparing botulinumtoxin formulations without the addition of animal-derived proteinaceousexcipients. In preferred embodiments, the formulations do not containany proteinaceous excipients at all. However, in certain embodiments,proteinaceous excipients not derived from animal sources (e.g.,recombinant albumin or recombinant gelatin) may be present in theformulations of the invention.

As used herein in connection with the botulinum toxin formulations ofthe invention, the term “stabilize” and variations thereof (e.g.,“stabilization”, “stabilizing” “stabilized” etc.) refers to theretention of biological activity of the botulinum toxin in theformulation over a specified time period as measured by a 20-mouse LD₅₀assay (See L B Pearce, R E Borodic, Tox. Appl. Pharmacol, v. 128, p. 69,1994 and ICCVAM/NICEATM/ECVAM, Scientific Workshop on Alternate Methodsto Refine, Reduce and Replace the Mouse LD50 Assay for Botulinum ToxinTesting, 14 Nov. 2006). In preferred embodiments, there is 100%retention of the biological activity of the botulinum toxin over thespecified time period. However, in other embodiments, there is at leasta 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% retention of thebiological activity of the botulinum toxin over the specified timeperiod. In certain embodiments, the duration of the specified timeperiod may be chosen to be consistent with the duration of the botulinumtoxin product manufacturing processes. For instance, the duration of thespecified time period may be chosen to be sufficient to stabilize thebotulinum toxin as it is subjected to one or more processing steps. Inother embodiments, the specified time will be on the order of weeks,months or even years, such as the case when the non-toxin formulationcomponents are selected to permit long-term stabilization of thebotulinum toxin formulation for storage. In certain embodiments, thespecified time is at least two weeks, at least one month, at least threemonths, at least six months, at least nine months, at least one year, atleast 18 months or at least two years. In other embodiments, thespecified time is coincident with the time for botulinum toxin to retaina desired amount of bioactivity using the formulations of the invention,which time includes, for example, the duration of the activities statedabove. The components used to stabilize the formulation may be selectedto permit stabilization at low temperatures (e.g., −5 to −10° C.) or atambient temperature, as described herein.

In some embodiments, the botulinum toxin formulations of the inventionare provided in solid form. By way of example only, the formulations maybe lyophilized or vacuum-dried to produce a solid formulation. When abotulinum toxin formulation of the invention is provided in solid form,and the specified time period over which stability of the botulinumformulation is measured is a week or less, the activity that is observedupon reconstitution is said to be the activity upon initial recovery. Incertain preferred embodiments of the invention, there is at least a 70%,80%, or 90% retention in the activity upon initial recovery. In otherembodiments, there is at least a 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, or 99% retention of activity upon initial recovery. In aparticularly preferred embodiment, the activity upon initial recovery isgreater than 99% and can even be 100%.

In other embodiments, the specified time over which stability ismeasured is greater than 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,or 24 months at given storage temperature. When a botulinum toxinformulation of the invention is provided in solid form, and thespecified time period over which stability of the botulinum formulationis measured twelve months or greater, the activity that is observed uponreconstitution is said to be the activity upon long term storage. Incertain preferred embodiments, there is at least a 70%, 80%, or 90%retention of botulinum toxin activity upon long term storage. In certainembodiments there is a 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or99% retention of botulinum toxin activity upon long term storage. Incertain embodiments, there is at least a 90% retention of botulinumtoxin activity after 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or24 months of storage at 25° C.; at least a 91% retention of botulinumtoxin activity after 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or24 months of storage at 25° C.; at least a 92% retention of botulinumtoxin activity after 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or24 months of storage at 25° C.; at least a 93% retention of botulinumtoxin activity after 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or24 months of storage at 25° C.; at least a 94% retention of botulinumtoxin activity after 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or24 months of storage at 25° C.; at least a 95% retention of botulinumtoxin activity after 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or24 months of storage at 25° C.; at least a 96% retention of botulinumtoxin activity after 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or24 months of storage at 25° C.; at least a 97% retention of botulinumtoxin activity after 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or24 months of storage at 25° C.; at least a 98% retention of botulinumtoxin activity after 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or24 months of storage at 25° C.; or at least a 99% retention of botulinumtoxin activity after 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or24 months of storage at 25° C.

In some embodiments of the invention, the botulinum toxin formulationsare stored in liquid form, rather than solid form. In certain preferredembodiments, the liquid botulinum toxin formulations according to theinvention retain at least 60%, 70%, 80%, or 90% of the botulinum toxinactivity for up to 8 hours at room temperature. In a particularlypreferred embodiment, liquid botulinum toxin formulations according tothe invention retain 100% of the botulinum toxin activity for up to 8hours at room temperature.

The excipients in the botulinum formulations of the inventionadvantageously reduce or eliminate mechanisms by which the botulinumtoxin is lost during manufacturing and storage. Without wishing to belimited by theory, it is believed that the non-proteinaceous excipientsof the invention reduce or eliminate unwanted adhesion of the botulinumtoxin to the vessels used for manufacturing, storage, or delivery of theformulation. Non-limiting examples of such vessels includes those madewith glass, polystyrene, polypropylene, and other polymers. Furthermore,and again without wishing to be limited by theory, it is believed thatthe non-proteinaceous excipients of the invention reduce or to preventunwanted reactions between the botulinum toxin and objects or substancesencountered by the botulinum toxin during manufacturing, storage, ordelivery. In preferred embodiments, the non-proteinaceous excipients areinert to the other components of the formulation.

In some embodiments, the botulinum toxin formulations are prepared in apowder form for ease of handling, transport, or storage. The powder formmay be prepared by any method known in the art. Non-limiting examples ofsuch methods include lyophilization, vacuum-drying, drum-drying andspray drying, with lyophilization and vacuum-drying being particularlypreferred.

The term “botulinum toxin” as used herein is meant to refer to any ofthe known types of botulinum toxin, whether produced by the bacterium orby recombinant techniques, as well as any such types that may besubsequently discovered including engineered variants or fusionproteins. As mentioned above, at the present time, seven immunologicallydistinct botulinum neurotoxins have been characterized, namely botulinumneurotoxin serotypes A, B, C, D, E, F and G, each of which isdistinguished by neutralization with type-specific antibodies. Thebotulinum toxin serotypes are available from Sigma-Aldrich and fromMetabiologics, Inc. (Madison, Wis.), as well as from other sources. Thedifferent serotypes of botulinum toxin vary in the animal species thatthey affect and in the severity and duration of the paralysis theyevoke.

The botulinum toxin used in the compositions of this invention canalternatively be a botulinum toxin derivative, that is, a compound thathas botulinum toxin activity but contains one or more chemical orfunctional alterations on any part or on any chain relative to naturallyoccurring or recombinant native botulinum toxins. For instance, thebotulinum toxin may be a modified neurotoxin (e.g., a neurotoxin whichhas at least one of its amino acids deleted, modified or replaced, ascompared to a native, or a recombinantly produced neurotoxin or aderivative or fragment thereof). For instance, the botulinum toxin maybe one that has been modified in a way that, for instance, enhances itsproperties or decreases undesirable side effects, but that still retainsthe desired botulinum toxin activity. The botulinum toxin may be any ofthe botulinum toxin complexes produced by the bacterium, as describedabove. Alternatively, the botulinum toxin may be a toxin prepared usingrecombinant or synthetic chemical techniques (e.g. a recombinantpeptide, a fusion protein, or a hybrid neurotoxin, as prepared fromsubunits or domains of different botulinum toxin serotypes (see U.S.Pat. No. 6,444,209, for instance)). The botulinum toxin may also be aportion of the overall molecule that has been shown to possess thenecessary botulinum toxin activity, and in such case may be used per seor as part of a combination or conjugate molecule, for instance a fusionprotein. Additionally, the botulinum toxin may be in the form of abotulinum toxin precursor, which may itself be non-toxic, for instance anontoxic zinc protease that becomes toxic on proteolytic cleavage.

The term “botulinum toxin complex” or “toxin complex” as used hereinrefers to the approximately 150 kD botulinum toxin protein molecule(belonging to any one of botulinum toxin serotypes A-G), along withassociated endogenous non-toxin proteins (i.e., hemagglutinin proteinand non-toxin non-hemagglutinin protein produced by Clostridiumbotulinum bacteria). Note, however, that the botulinum toxin complexneed not be derived from Clostridium botulinum bacteria as one unitarytoxin complex. For example, botulinum toxin or modified botulinum toxinmay be recombinantly prepared first and then subsequently combined withthe non-toxin proteins. Recombinant botulinum toxin can also bepurchased (e.g., from List Biological Laboratories, Campbell, Calif.)and then combined with non-toxin proteins.

This invention also contemplates “reduced botulinum toxin complexes,” inwhich the botulinum toxin complexes have reduced amounts of non-toxinprotein compared to the amounts naturally found in botulinum toxincomplexes produced by Clostridium botulinum bacteria. In one embodiment,reduced botulinum toxin complexes are prepared using any conventionalprotein separation method to extract a fraction of the hemagglutininprotein or non-toxin non-hemagglutinin protein from botulinum toxincomplexes derived from Clostridium botulinum bacteria. For example,reduced botulinum toxin complexes may be produced by dissociatingbotulinum toxin complexes through exposure to red blood cells at a pH of7.3 (e.g., see EP 1514556 A1, hereby incorporated by reference). HPLC,dialysis, columns, centrifugation, and other methods for extractingproteins from proteins can be used. Alternatively, when the reducedbotulinum toxin complexes are to be produced by combining syntheticallyproduced botulinum toxin with non-toxin proteins, one may simply addless hemagglutinin or non-toxin non-hemagglutinin protein to the mixturethan what would be present for naturally occurring botulinum toxincomplexes. Any of the non-toxin proteins (e.g., hemagglutinin protein ornon-toxin non-hemagglutinin protein or both) in the reduced botulinumtoxin complexes according to the invention may be reduced independentlyby any amount. In certain exemplary embodiments, one or more non-toxinproteins are reduced by at least about 0.5%, 1%, 3%, 5%, 10%, 20%, 30%,40%, 50%, 60%, 70%, 80%, or 90% compared to the amounts normally foundin botulinum toxin complexes. In one embodiment, substantially all ofthe non-toxin protein (e.g., >95% of the hemagglutinin protein andnon-toxin non-hemagglutinin protein) that would normally be found inbotulinum toxin complexes derived from Clostridium botulinum bacteria isremoved from the botulinum toxin complex. In another embodiment, onlythe pure botulinum toxin molecule, without any hemagglutinin protein andnon-toxin non-hemagglutinin protein, is used. Furthermore, although theamount of endogenous non-toxin proteins may be reduced by the sameamount in some cases, this invention also contemplates reducing each ofthe endogenous non-toxin proteins by different amounts, as well asreducing at least one of the endogenous non-toxin proteins, but not theothers.

This invention also contemplates the general use of combinations andmixtures of botulinum toxins, although due to their differing nature andproperties, mixtures of botulinum toxin serotypes are not generallyadministered at this time in the health-care or cosmetic industries.

The botulinum toxin formulations of the invention comprise a non-ionicsurfactant. Generally, this invention contemplates the use of anynon-ionic surfactant that has the ability to stabilize botulinum toxinand that is suitable for pharmaceutical use. In certain embodiments, thenon-ionic surfactant is a polysorbate, non-limiting examples of whichinclude polysorbate 20, polysorbate 40, polysorbate 60, and polysorbate80. In other embodiments, the non-ionic surfactant is a sorbitan ester,non-limiting examples of which include SPAN® 20, SPAN® 60, SPAN® 65, andSPAN® 80. The invention also contemplates the use of poloxamers,non-limiting examples of which include poloxamer 181, poloxamer 188, andpoloxamer 407. The invention also contemplates using TRITON™ X-100 orNP-40 as the non-ionic surfactants. In addition, the inventioncontemplates embodiments in which combinations of different non-ionicsurfactants are used in conjunction. In certain preferred embodiments,the non-ionic surfactant is selected from the group consisting ofpolysorbates, poloxamers, and sorbitans, with polysorbates and sorbitansbeing particularly preferred. In preferred embodiments, theconcentration of the non-ionic surfactant is in the range of 0.005% to0.5%, or in the range of 0.01% to 0.2%, or in the range of 0.02% to 0.1%or in the range of 0.05 to 0.08%. This invention also contemplatesformulations where the concentration of the non-ionic surfactant is0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.10%,0.11%, 0.12%, 0.13%, 0.14%, or 0.15%.

The botulinum toxin formulations of the invention comprise anon-reducing sugar. In preferred embodiments, the non-reducing sugar hasa glass transition temperature above 55° C., 57° C., or 60° C. Withoutwishing to be bound by theory, it is believed that such glass transitiontemperatures are sufficiently high to suppress undesirable molecularmotions that cause the botulinum toxin to denature. In certainparticularly preferred embodiments, the non-reducing sugar is adisaccharide, non-limiting examples of which include trehalose andsucrose. In other embodiments, the non-reducing sugar is atrisaccharide, a non-limiting example of which is raffinose. Generally,the concentration of the non-reducing sugar in the botulinum toxinformulations of the invention are in the range of 10% to 40% (w/v),preferably 10% to 25% (w/v), more preferably 15% to 20% (w/v). In somepreferred embodiments, the concentration of the non-reducing sugar is10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19% or 20% (w/v). When thenon-reducing sugar is trehalose, preferably the hydrated form oftrehalose (i.e., trehalose-dihydrate) is used to prepare theformulation, although use of the anhydrous form of trehalose iscontemplated as well.

Furthermore, one aspect of this invention is the recognition that thechoice of non-reducing sugar may be used to tailor the stability of thebotulinum toxin formulation as a function of temperature. For instance,when the botulinum toxin formulation is subjected to conditions withoutrefrigeration it is advantageous to use trehalose as the non-reducingsugar, because trehalose confers stability to the botulinum toxin atambient temperatures. Thus, trehalose-containing formulations of theinvention may be advantageously processed at ambient temperatures duringvarious manufacturing processes without appreciable loss of botulinumtoxin activity. In situations where the botulinum toxin formulation willbe refrigerated (e.g., during long term storage lasting months or evenyears), one may choose sucrose as the non-reducing sugar. Mixtures ofsugars are also contemplated by the invention. For example, in certainembodiments, both sucrose and trehalose are added to the botulinum toxinformulation. The specific ratio of components of the sugar mixture willdepend upon the effect to be achieved and can be determined by routineexperimentation. Also within the scope of this invention is the use ofdifferent non-reducing sugars, such as disaccharides, alone or incombination during different stages of purification, manufacturing, orstorage. Thus, for example, trehalose may be used during initialprocessing and/or purification at higher temperatures (e.g., ambienttemperature), followed by removal and replacement of the trehalose withsucrose. The removal and substitution of one non-reducing sugar, such asa disaccharide, for another may be accomplished by, for example,dialysis, chromatography, or other methods known in the art.

Another aspect of the invention is the recognition that the non-reducingsugar (or sugars) specified herein may, for certain embodiments, act asthe primary bulking agent for solid botulinum toxin formulations. Inparticular, it has been discovered that the non-reducing sugars, whenadded in the amounts specified herein, can form a stable andmechanically robust cake upon lyophilization or vacuum-drying of thebotulinum toxin formulation. Without wishing to be limited by theory, itis believed that the non-reducing sugars in such formulations form anamorphous solid in which the hydroxyl groups on the non-reducing sugarrandomly orient the non-reducing sugar to maximize hydrogen bonding,which stabilizes the botulinum toxin and imparts mechanical robustnessto the solid cake. In some preferred embodiments, the non-reducingsugars described herein are used as the sole bulking agent for thebotulinum toxin formulations. In other preferred embodiments, anon-reducing sugar acts as the primary bulking agent, but small amountsof secondary bulking agents are added to the botulinum toxinformulation. The secondary bulking agents are not particularly limited,and may be any bulking agent that does not have an unacceptably adverseeffect on the stability of the botulinum toxin formulation. When usingsecondary bulking agents that have a tendency to crystallize, it ispreferable to add them in sufficiently low concentrations, so that theyare unable to crystallize. Without wishing to be limited by theory, itis believed that crystallization of such secondary bulking agents wouldhave an adverse effect on the stability of the solid cake obtained afterlyophilization or vacuum-drying.

A further aspect of the invention is the unexpected result thatnon-reducing sugars, at the concentrations described herein, may be usednot only to stabilize lyophilized botulinum toxin formulations, but alsobotulinum toxin formulations in the solution phase.

In preferred embodiments, the botulinum toxin formulation comprises abuffer. Generally, any physiologically compatible buffer capable ofmaintaining the pH in the range of 4.5 to 6.5, more preferably in therange of 5 to 6, and most preferably about 5.5, is suitable for thebotulinum toxin formulations of the invention. Non-limiting examples ofsuch buffers include those involving salts of citric acid, acetic acid,succinic acid, tartaric acid, maleic acid, and histidine. Non-limitingexamples of suitable buffer concentrations include buffer concentrationsin the range of 0.400% to 0.600%; 0.450% to 0.575%, or 0.500% to 0.565%.The invention also contemplates botulinum toxin formulations comprisinga mixture of buffer salts, non-limiting examples of which includecitrate/acetate, citrate/histidine, citrate/tartrate, maleate/histidine,or succinate/histidine. In certain preferred embodiments, the buffer isphosphate buffer.

In certain embodiments, the botulinum toxin formulations comprise abulking agent (in addition to the non-reducing sugar(s)) which makes iteasier to handle lyophilized forms of the botulinum toxin formulations.In certain preferred embodiments, the bulking agents crystallize underlyophilization conditions and do not mix well with the other excipientswhen in the solid state. However, in other preferred embodiments, thebulking agent remains amorphous under lyophilization conditions, even ifit is known to have a tendency to crystallize during freezing (e.g.,mannitol or glycine). As will be understood by a person of ordinaryskill in the art, whether a bulking agent will crystallize or remainamorphous during lyophilization is governed by several factors,including the type of bulking agent, the amount of bulking agentrelative to the other components of the formulation, and the rate atwhich the formulation is lyophilized. In certain preferred embodiments,the amount of non-reducing sugar relative to bulking agent is adjustedto suppress the crystallization of the bulking agent. When this is thecase, the ratio of non-reducing sugar to bulking agent, on a weightpercent basis, may be greater than 0.33 to 1; 0.5 to 1, 0.75 to 1; 1:1;2:1, 3:1, or 4:1. Non-limiting examples of bulking agents contemplatedby the invention include sorbitol, mannitol, glycine, arginine, andhistidine. In some embodiments, the concentration of the bulking agentmay be in the range of 1% to 10%, 2% to 6%, 3% to 5% or 4% to 4.5%(w/v). In certain preferred embodiments, when a bulking agent is used,the concentration of the non-reducing sugar may be reduced from the 10%to 40% (w/v) range to a range of 0.5% to 3.0% (w/v). Furthermore, incertain preferred embodiments, the ratio of the non-reducing sugar tothe bulking agent is in the range of 0.07 to 2.0, preferably in therange of 0.4 to 0.6. Thus, by way of example only, the formulation maycomprise mannitol as the bulking agent and trehalose dihydrate as thenon-reducing sugar, with mannitol present in a concentration range of1.5% to 7.5% (w/v) and trehalose dihydrate present in a concentrationrange of 0.5% to 3.0% (w/v). Preferably, the bulking agent is not sodiumchloride.

The botulinum toxin formulations of the invention can be administeredvia any conventional route of administration. In some embodiments of theinvention, the botulinum toxin formulations are administered byintramuscular or subcutaneous injection into to the subject. In otherembodiments, the botulinum toxin formulations are administeredtopically. Regardless of whether the botulinum toxin formulations areadministered topically or by injection, the formulations may include apositively charged carrier having positively charged efficiency groups,as described herein, in order to facilitate the penetration of thebotulinum toxin into the issues of interest. It should be noted,however, that the positively charged carrier is not required forstabilization and that the invention contemplates embodiments in whichthe formulations do not comprise such a positively charged carrier. Thebotulinum toxin formulations of the invention may also be administeredvia a drug delivery device, a non-limiting example of which is a skinpatch.

When the botulinum toxin formulations are to be administered topically,it is advantageous to include in the formulations a positively chargedcarrier with positively charged branching groups, as described herein,to promote transdermal penetration of the botulinum toxin (see also U.S.patent application Ser. Nos. 09/910,432; 11/073,307; 11/072,026; andSer. No. 10/793,138, each of which is incorporated by reference in itsentirety). Without the use of the positively charged carrier or someother means of enhancing transdermal transport, the transdermal flux oftopically applied botulinum toxin would be expected to be quite low. Itshould be noted that the invention also contemplates the use of othermethods of enhancing transdermal transport, besides the use of apositively charge carrier as described herein, with the botulinum toxinformulations of the invention. Non-limiting examples of such methodsinclude transdermal delivery of the botulinum toxin formulations usingliposomes, iontophoresis, micelles, and the like.

Furthermore, when the botulinum toxin formulations are to beadministered topically, it is often advantageous to mix them withgelling agents and/or viscosity-modifying agents to increase theviscosity of the formulation, order make the application of thebotulinum toxin easier and more accurate. Additionally, if a positivelycharged carrier is used, these agents help to prevent the aqueousbotulinum toxin/carrier formulation from drying out, which tends tocause a decrease in the activity of the botulinum toxin. Particularlypreferred agents are those that are uncharged and do not interfere withthe botulinum toxin activity or the efficiency of the toxin-carriercomplexes in crossing skin. The gelling agents may be certaincellulose-based gelling agents, such as hydroxypropylcellulose (HPC) forexample. In some embodiments, the botulinum toxin/carrier complex isformulated in a composition having 2-4% HPC. Alternatively, theviscosity of a solution containing a botulinum toxin/carrier complex maybe altered by adding polyethylene glycol (PEG). In other embodiments,the botulinum toxin/carrier solution is combined with pre-mixed viscousagents, such as Cetaphil® moisturizer. In other embodiments, theviscosity modifying agent is a poloxamer, non-limiting examples of whichinclude poloxamer 188 and poloxamer 407. The viscosity-modifying agentmay also be a polyacrylic acid, a polyamide, or a vegetable gum (e.g.,guar gum). Furthermore, botulinum toxin formulations as described hereinmay be mixed with excipients to form emulsions (includingmicroemulsions), suspensions, creams, lotions, gels, powders, or othertypical solid or liquid compositions used for application to skin andother tissues where the compositions may be used. Such compositions mayalso contain other ingredients typically used in such products, such asantimicrobials, moisturizers and hydration agents, penetration agents,preservatives, emulsifiers, natural or synthetic oils, solvents,surfactants, detergents, emollients, antioxidants, fragrances, fillers,thickeners, waxes, odor absorbers, dyestuffs, coloring agents, powders,and optionally anesthetics, anti-itch additives, botanical extracts,conditioning agents, darkening or lightening agents, glitter,humectants, mica, minerals, polyphenols, silicones or derivativesthereof, sunblocks, vitamins, and phytomedicinals.

This invention also provides kits for preparing and/or administering thebotulinum toxin formulations of the invention. In some embodiments, thekit comprises the botulinum toxin, as well as such additional excipientsthat are needed to produce a stabilized formulation according to theinvention. The kit may also comprise a premix that may in turn be usedto produce such a formulation. In other embodiments, the kit comprises abotulinum toxin formulation according to the invention that has beenlyophilized and a device for delivering the formulation, a non-limitingexample of which is a syringe.

The invention provides formulations that deliver a “therapeutic amount”of botulinum to the subject during treatment. As used herein, the term“therapeutic amount” refers to an amount of botulinum toxin that issufficient to produce the desired effect (e.g., relaxation of muscles,treatment of wrinkles, treatment of pain, or reduction of activity of anoveractive gland, such as a sweat gland). The “therapeutic amount” ofbotulinum toxin is implicitly understood to be a safe amount that doesnot cause unwanted paralysis or other undesired or harmful side effects.The specific amount of botulinum toxin that is administered will dependon several factors, including the route of administration, the site ofadministration, the indication to be treated, and the serotype (orserotypes) of botulinum toxin in the specific formulation. For instance,when the type A serotype botulinum toxin is selected, a therapeuticamount may be in the range of 10 U to 150 U or 1,000 U to 2,500 U. Inother preferred embodiments, the therapeutic amount ranges from 400 U to800 U and 1,000 U to 50,000 U, preferably from 2,000-35,000 U, morepreferably from 3,000 U-30,000 U, and even more preferably from 4,000 Uto 25,000 U. In certain embodiments, the therapeutic amount ranges from4,000 U to 8,000 U, 9,000 U to 19,000 U, or 20,000 U to 40,000 U.

The compositions of the invention are applied so as to cause a desiredeffect, which may be a cosmetic or a therapeutic effect. Desired effectsinclude the relaxation of certain muscles with the aim of, for instance,decreasing the appearance of fine lines and/or wrinkles, especially inthe face, or adjusting facial appearance in other ways such as wideningthe eyes, lifting the corners of the mouth, or smoothing lines that fanout from the upper lip, or the general relief of muscular tension. Thelast-mentioned effect, general relief of muscular tension, can beeffected in the face or elsewhere. The compositions of the invention maycontain an appropriate effective amount of the botulinum toxin forapplication as a single-dose treatment, or may be more concentrated,either for dilution at the place of administration or for use inmultiple applications. The stabilized botulinum toxin complexes orstabilized reduced botulinum toxin complexes can be administered to asubject for treating conditions such as undesirable facial muscle orother muscular spasms, hyperhidrosis, acne, or conditions elsewhere inthe body in which relief of muscular ache or spasms is desired. Thebotulinum toxin is administered delivery to muscles or to otherskin-associated structures. The administration may be made, for example,to the face, legs, shoulders, back (including lower back), axilla,palms, feet, neck, groin, dorsa of the hands or feet, elbows, upperarms, knees, upper legs, buttocks, torso, pelvis, or any other part ofthe body where administration of the botulinum toxin is desired.

In certain preferred embodiments, the botulinum toxin formulations ofthe invention comprise a positively charged carrier with positivelycharged efficiency groups. When this is the case, the positively chargedcarrier is present in an amount sufficient to facilitate penetration ofthe botulinum toxin into the tissues of interest. As used herein, theterm “positively charged” means that the carrier has a positive chargeunder at least some solution-phase conditions, more preferably under atleast some physiologically compatible conditions. More specifically,“positively charged” means that the group in question containsfunctionalities that are charged under all pH conditions, for instance,a quaternary amine, or contains a functionality which can acquirepositive charge under certain solution-phase conditions, such as pHchanges in the case of primary amines. More preferably, “positivelycharged” as used herein refers to those groups that have the behavior ofassociating with anions over physiologically compatible conditions.Polymers with a multiplicity of positively-charged moieties need not behomopolymers, as will be apparent to one skilled in the art. Otherexamples of positively charged moieties are well known in the prior artand can be employed readily, as will be apparent to those skilled in theart.

Generally, the positively-charged carrier comprises a positively chargedbackbone, which is typically a chain of atoms, either with groups in thechain carrying a positive charge at physiological pH, or with groupscarrying a positive charge attached to side chains extending from thebackbone. Preferably, the positively charged backbone itself will nothave a defined enzymatic or therapeutic biologic activity. The linearbackbone is a hydrocarbon backbone which is, in some embodiments,interrupted by heteroatoms selected from nitrogen, oxygen, sulfur,silicon and phosphorus. The majority of backbone chain atoms are usuallycarbon. Additionally, the backbone will often be a polymer of repeatingunits (e.g., amino acids, poly(ethyleneoxy), poly(propyleneamine),polyalkyleneimine, and the like) but can be a heteropolymer. In onegroup of embodiments, the positively charged backbone is apolypropyleneamine wherein a number of the amine nitrogen atoms arepresent as ammonium groups (tetra-substituted) carrying a positivecharge. In another embodiment, the positively charged backbone is anonpeptidyl polymer, which may be a hetero- or homo-polymer such as apolyalkyleneimine, for example a polyethyleneimine orpolypropyleneimine, having a molecular weight of from about 100 to about2,500,000 D, preferably from about 250 to about 1,800,000 D, and mostpreferably from about 1000 to about 1,400,000 D. In another group ofembodiments, the backbone has attached a plurality of side-chainmoieties that include positively charged groups (e.g., ammonium groups,pyridinium groups, phosphonium groups, sulfonium groups, guanidiniumgroups, or amidinium groups). The sidechain moieties in this group ofembodiments can be placed at spacings along the backbone that areconsistent in separations or variable. Additionally, the length of thesidechains can be similar or dissimilar. For example, in one group ofembodiments, the sidechains can be linear or branched hydrocarbon chainshaving from one to twenty carbon atoms and terminating at the distal end(away from the backbone) in one of the above-noted positively chargedgroups. In all aspects of the present invention, the association betweenthe carrier and the botulinum toxin is by non-covalent interaction,non-limiting examples of which include ionic interactions, hydrogenbonding, van der Waals forces, or combinations thereof.

In one group of embodiments, the positively charged backbone is apolypeptide having multiple positively charged sidechain groups (e.g.,lysine, arginine, ornithine, homoarginine, and the like). Preferably,the polypeptide has a molecular weight of from about 100 to about1,500,000 D, more preferably from about 250 to about 1,200,000 D, mostpreferably from about 1000 to about 1,000,000 D. One of skill in the artwill appreciate that when amino acids are used in this portion of theinvention, the sidechains can have either the D- or L-form (R or Sconfiguration) at the center of attachment. In certain preferredembodiments, the polypeptide has a molecular weight from about 500 toabout 5000 D, more preferably from 1000 to about 4000 D, more preferablyfrom 2000 to about 3000 D. In other embodiments, the polypeptide has amolecular weight of at least about 10,000.

In another embodiment, the backbone portion is a polylysine andefficiency groups, as discussed herein, are attached to the polylysine.The polylysine may have a molecular weight of from about 100 to about1,500,000 D, preferably from about 250 to about 1,200,000 D, and mostpreferably from about 1000 to about 3000 D. In one exemplary embodiment,the positively charged carrier with positively charged efficiency groupsis a peptide with the amino acid sequence RKKRRQRRR-G-(K)₁₅-G-RKKRRQRRR(SEQ ID NO: 1), RGRDDRRQRRR-G-(K)₁₅-G-RGRDDRRQRRR (SEQ ID NO: 2), orYGRKKRRQRRR-G-(K)₁₅-G-YGRKKRRQRRR (SEQ ID NO: 3). It also can be any ofthe commercially available (Sigma Chemical Company, St. Louis, Mo., USA)polylysines such as, for example, polylysine having MW>70,000 D,polylysine having MW of 70,000 to 150,000 D, polylysine having MW150,000 to 300,000 D and polylysine having MW>300,000 D. The selectionof an appropriate polylysine will depend on the remaining components ofthe composition and will be sufficient to provide an overall netpositive charge to the composition and provide a length that ispreferably from one to four times the combined length of the negativelycharged components.

Alternatively, the backbone can be an analog of a polypeptide such as apeptoid. See, for example, Kessler, Angew. Chem. Int. Ed. Engl. 32:543(1993); Zuckermann et al. Chemtracts—Macromol. Chem. 4:80 (1992); andSimon et al. Proc. Nat'l. Acad. Sci. USA 89:9367 (1992)). Briefly, apeptoid is a polyglycine in which the sidechain is attached to thebackbone nitrogen atoms rather than the alpha-carbon atoms. As above, aportion of the sidechains will typically terminate in a positivelycharged group to provide a positively charged backbone component.Synthesis of peptoids is described in, for example, U.S. Pat. No.5,877,278, which is hereby incorporated by reference in its entirety. Asthe term is used herein, positively charged backbones that have apeptoid backbone construction are considered “non-peptide” as they arenot composed of amino acids having naturally occurring sidechains at theα-carbon locations.

A variety of other backbones can be used employing, for example, stericor electronic mimics of polypeptides wherein the amide linkages of thepeptide are replaced with surrogates such as ester linkages, thioamides(—CSNH—), reversed thioamide (—NHCS—), aminomethylene (—NHCH₂—) or thereversed methyleneamino (—CH₂NH—) groups, keto-methylene (—COCH₂—)groups, phosphinate (—PO₂RCH₂—), phosphonamidate and phosphonamidateester (—PO₂RNH—), reverse peptide (—NHCO—), trans-alkene (—CR═CH—),fluoroalkene (—CF═CH—), dimethylene (—CH₂CH₂—), thioether (—CH₂S—),hydroxyethylene (—CH(OH)CH₂—), methyleneoxy (—CH₂O—), tetrazole (CN₄),sulfonamido (—SO₂NH—), methylenesulfonamido (—CHRSO₂NH—), reversedsulfonamide (—NHSO₂—), and backbones with malonate and/orgem-diamino-alkyl subunits, for example, as reviewed by Fletcher et al.((1998) Chem. Rev. 98:763) and detailed by references cited therein.Many of the foregoing substitutions result in approximately isostericpolymer backbones relative to backbones formed from α-amino acids.

In each of the backbones provided above, sidechain groups can beappended that carry a positively charged group. For example, thesulfonamide-linked backbones (—SO₂NH— and —NHSO₂—) can have sidechaingroups attached to the nitrogen atoms. Similarly, the hydroxyethylene(—CH(OH)CH₂—) linkage can bear a sidechain group attached to the hydroxysubstituent. One of skill in the art can readily adapt the other linkagechemistries to provide positively charged sidechain groups usingstandard synthetic methods.

In some embodiments of the invention, the positively charged carriercomprises positively charged efficiency groups. Non-limiting examples ofefficiency groups include -(gly)_(n1)-(arg)_(n2) (SEQ ID NO: 4), inwhich the subscript n1 is an integer of from 0 to 20 more preferably 0to 8, still more preferably 2 to 5, and the subscript n2 isindependently an odd integer of from about 5 to about 25, morepreferably from about 7 to about 17, and most preferably from about 7 toabout 13, HIV-TAT or fragments thereof, or Antennapedia PTD or afragment thereof. Preferably the side-chain or branching groups have thegeneral formula -(gly)_(n1)-(arg)_(n2)(SEQ ID NO: 4), as describedabove. Other preferred embodiments are those in which the branching orefficiency groups are HIV-TAT fragments that have the formula(gly)_(p)-RGRDDRRQRRR-(gly)_(q) (SEQ ID NO: 5),(gly)_(p)-YGRKKRRQRRR-(gly)_(q) (SEQ ID NO: 6), or(gly)_(p)-RKKRRQRRR-(gly)_(q) (SEQ ID NO: 7), wherein the subscripts pand q are each independently an integer of from 0 to 20 and the fragmentis attached to the carrier molecule via either the C-terminus or theN-terminus of the fragment. The side branching groups can have eitherthe D- or L-form (R or S configuration) at the center of attachment.Preferred HIV-TAT fragments are those in which the subscripts p and qare each independently integers of from 0 to 8, more preferably 2 to 5.Other preferred embodiments are those in which the branching groups areAntennapedia PTD groups or fragments thereof that retain the group'sactivity. These are known in the art, for instance, from Console et al.,J. Biol. Chem. 278:35109 (2003). Preferably, the positively chargedcarrier includes side-chain positively charged branching groups in anamount of at least about 0.05%, as a percentage of the total carrierweight, preferably from about 0.05 to about 45 weight %, and mostpreferably from about 0.1 to about 30 weight %. For positively chargedbranching groups having the formula -(gly)_(n1)-(arg)_(n2) (SEQ ID NO:4), the most preferred amount is from about 0.1 to about 25%.

The following examples are meant to provide non-limiting illustrationsof various embodiments of the invention. As a person of ordinary skillin the art will recognize, modifications may be made without departingfrom the spirit and the scope of the invention.

EXAMPLES Example 1: Preparation of a Botulinum Toxin Formulation

An exemplary botulinum toxin formulation of the invention was preparedby combining appropriate amounts of trehalose dihydrate, polysorbate-20,histidine, and histidine HCl to produce a 2× formulation stock solutionthat contained 36% trehalose dihydrate, 0.05% polysorbate-20, and 1.126%histidine at a pH of 5.5. The solution was cooled to 4° C. Botulinumtoxin API was pelleted by centrifugation from an ammonium sulfate stocksuspension. The toxin pellet was dissolved in 0.56% histidine buffer pH5.5 with 0.05% polysorbate-20. This solution was further diluted withthe histidine/polysorbate-20 solution to give a toxin stock solution of1.074 μg/mL solution.

A stock solution of the carrier peptide RKKRRQRRR-G-(K)₁₅-G-RKKRRQRRR(SEQ ID NO: 1), was prepared by dissolving sufficient peptide into waterto produce a solution of 6.0 mg/mL peptide. Toxin stock solution,peptide stock solution, 0.56% histidine buffer, and 2× formulation stocksolution were combined to produce a formulation bulk drug productconsisting of 18% trehalose dihydrate, 0.025% polysorbate-20, 0.56%histidine buffer, 55 ng/mL toxin and 150 μg/mL carrier peptide.

Example 2: Lyophilization of a Botulinum Toxin Formulation

This example provides a lyophilization procedure for a botulinum toxinformulation according to the invention. 200 μL aliquots of theformulation bulk drug product described in Example 1 was transferred toeach of fifty-five 2-mL glass vials. Grey butyl rubber lyophilizationstoppers were loosely placed on top of the glass vials. The vials wereplaced into the lyophilizer and lyophilization initiated.

The lyophilization process comprised three main stages: (1) a freezingstage; (2) a primary drying stage; and (3) a secondary drying stage.Each of these three main stages contained one or more individual processsteps, which were performed at the temperatures and pressures asindicated below. The primary drying time required to complete primarydrying varied depending upon the number of vials and the fill volume inthe vials.

Freezing Step Rate/Hold deg C. Minutes 1 H 5 50 2 R −45 60 3 H −45 120Freeze, Condenser and Evacuate: Freeze Temperature: −40 deg C. Shelf orProduct Ave Extra Freeze Time: 0 minutes Vacuum Start Permit: −45 AvgCond deg C. Heat Start Permit: 200 mTorr Primary Drying Step Rate/Holddeg C. Minutes mTorr 1 H −45 150 150 2 H −33 2520 150 3 R −25 990 150 4H −25 275 150 5 H −15 240 150 6 H 0 300 150 7 R 25 180 150 8 H 25 120150 Secondary Drying Step 1 H 27 160 100

Example 3: Stability of Botulinum Toxin Formulation with TrehaloseDihydrate

FIGS. 1 and 2 show the results of stability studies in which vials ofthe botulinum toxin formulation prepared at two different toxinconcentrations with trehalose dihydrate (11 ng/vial and 1.1 ng/vial)were prepared as described above, stored at 4° C., 25° C. and 40° C. andtested at the indicated time points to determine the biological activityof the botulinum toxin for a duration of up to 18 months. The activitywas measured using the LD₅₀ mouse assay and the activities are reportedin FIGS. 1 and 2 as equivalent units of botulinum toxin per vial. At thebeginning of the experiment (t=0), the observed activity was 1802 unitsof botulinum toxin per vial for the formulation in FIG. 1 and 192 unitsof botulinum toxin per vial for the formulation in FIG. 2. The observedvariability in the botulinum toxin activity in FIGS. 1 and 2 is believedto result from the inherent noise in the data obtained using the LD₅₀mouse assay and the small sample number (n=1vial/temperature-timepoint). Despite this variability, the data indicatethat there is no trend to loss of recovered activity over thiseighteen-month study, even at a storage temperature of 40° C. These dataprovide an example of the stabilization of an exemplary botulinum toxinformulation of the invention.

Example 4: Stability of Botulinum Toxin Formulation with Sucrose

FIG. 3 shows the results of stability studies in which vials of thebotulinum toxin formulation with sucrose at 11 ng/vial were preparedusing the protocol described above for the trehalose-containingformulations, stored at 4° C. and 25° C. and tested at the indicatedtime points to determine the biological activity of the botulinum toxinfor a duration of up to 18 months. The activity was measured using theLD₅₀ mouse assay as described above and the activities are reported inFIG. 3 as equivalent units of botulinum toxin per vial. At the beginningof the experiment (t=0), the observed activity was 2299 units ofbotulinum toxin per vial. The observed variability in the botulinumtoxin activity in FIG. 3 is believed to result from the inherent noisein the data obtained using the LD₅₀ mouse assay and the small samplenumber (n=1 vial/temperature-timepoint). Despite this variability, thedata indicate that there is no trend to loss of recovered activity overthis eighteen-month study, even at a storage temperature of 25° C. Thesedata provide an example of the stabilization of an exemplary botulinumtoxin formulation of the invention.

Example 5: Lyophilization of a Botulinum Toxin Formulation with BulkingAgents

In this example, the botulinum toxin formulation was prepared bycombining appropriate amounts of trehalose dihydrate, mannitol,polysorbate-20, histidine, and histidine HCl to produce a formulationstock solution that contained 3% trehalose dihydrate, 7.5% mannitol,0.05% polysorbate-20, and 0.563% histidine at a pH of 5.5. The solutionwas cooled to 4° C. Botulinum toxin API was pelleted by centrifugationfrom an ammonium sulfate stock suspension. The toxin pellet wasdissolved in the formulation stock solution. The concentration of thissolution was 117 μg/mL. This solution was further diluted with the samesolution to a final toxin stock solution concentration of 11.7 μg/mL. Asufficient amount of a carrier peptide having the amino acid sequenceRKKRRQRRR-G-(K)₁₅-G-RKKRRQRRR (SEQ ID NO: 1) was dissolved in water toprepare a 20 mg/mL carrier peptide stock solution. These solutions werecombined to create a formulation bulk drug product with a finalcomposition of 3% trehalose dihydrate, 7.5% mannitol, 0.05%polysorbate-20, and 0.563% histidine pH of 5.5, 75 μg/mL carrierpeptide, and 5.5 ng/mL toxin,

Aliquots of 200 μL of the formulation bulk drug product were added toeach of 192 2-mL glass lyophilization vials. Grey Butyl rubber stopperswere placed loosely on top of the vials. The vials were then placed inthe lyophilizer and lyophilization was initiated.

As in the previous example, the lyophilization process comprised threemain stages: (1) a freezing stage; (2) a primary drying stage; and (3) asecondary drying stage. Each of these three main stages contained one ormore individual process steps, which were performed at the temperaturesand pressures as indicated below:

Freezing Step Rate/Hold deg C. Minutes 1 H 5 30 2 R −45 50 3 H −45 60 4R −20 25 5 H −20 240 6 R −45 25 7 H −45 60 Freeze, Condenser andEvacuate: Freeze Temperature: −45 deg C. Shelf or Product Ave ExtraFreeze Time: 1 minutes Vacuum Start Permit: −40 Avg Cond deg C. HeatStart Permit: 300 mTorr Primary Drying Step Rate/Hold deg C. MinutesmTorr 1 H −45 30 150 2 R −28 80 150 3 H −28 900 150 4 R −15 20 150 5 H−15 180 150 6 R −5 20 150 7 H −5 180 150 8 R 27 100 150 Secondary DryingStep 1 H 27 540 100

What is claimed is:
 1. A method for stabilizing a botulinum toxincomposition comprising a botulinum toxin, a non-reducing disaccharide ora non-reducing trisaccharide, a physiologically compatible buffer, and anon-ionic surfactant, the method comprising: combining the botulinumtoxin, the non-reducing disaccharide or the non-reducing trisaccharide,the non-ionic surfactant, and a physiologically compatible buffer toform a liquid composition; wherein the concentration of the non-reducingdisaccharide or the non-reducing tri-saccharide present in the liquidcomposition is in the range of 10% to 40% (w/v), wherein theconcentration of the botulinum toxin present in the liquid compositionis not greater than 1.074 μg/mL; and drying the liquid composition toproduce a solid amorphous composition.
 2. The method according to claim1, wherein the pH of the liquid composition is in the range of pH 4.5 to6.5.
 3. The method according to claim 1, wherein the step of dryingcomprises lyophilizing or vacuum-drying the liquid composition toproduce the solid composition.
 4. The method according to claim 3,wherein the step of drying comprises lyophilization to produce the solidcomposition.
 5. The method according to claim 1, wherein the combiningstep is performed without adding animal-derived proteinaceousexcipients.
 6. The method according to claim 5, wherein the combiningstep is performed without adding animal-derived proteinaceous excipientscomprising albumin.
 7. The method according to claim 1, furthercomprising combining with the botulinum toxin, the non-reducingdisaccharide or the non-reducing trisaccharide, the non-ionicsurfactant, and the physiologically compatible buffer a positivelycharged backbone selected from (a) a positively charged peptide with anamino acid sequence selected from RKKRRQRRR-G-(K)15-G-RKKRRQRRR (SEQ IDNO: 1), RGRDDRRQRRR-G-(K)15-G-RGRDDRRQRRR (SEQ ID NO: 2), orYGRKKRRQRRR-G-(K)15-G-YGRKKRRQRRR (SEQ ID NO: 3); or (b) a positivelycharged polypeptide or a nonpeptidyl polymer having covalently attachedthereto at least one positively charged efficiency group having an aminoacid sequence selected from (gly)n1-(arg) n2 (SEQ ID NO: 4), wherein thesubscript n1 is an integer of from 0 to 20 and the subscript n2 isindependently an odd integer of from 5 to 25; (gly)p-RGRDDRRQRRR-(gly)q(SEQ ID NO 5); (gly)p-YGRKKRRQRRR-(gly)q (SEQ ID NO 6); (gly)p-RKKRRQRRR-(gly)q, (SEQ ID NO 7); wherein the subscripts p and q areeach independently an integer of from 0 to 20; to form the liquidcomposition comprising a formulation bulk drug product.
 8. The methodaccording to claim 7, wherein the positively charged backbone is apositively charged polypeptide which is polylysine.
 9. The methodaccording to claim 7, wherein the positively charged backbone is apositively charged nonpeptidyl polymer which is a polyalkyleneimine. 10.The method according to claim 8, wherein the non-reducing disaccharideor tri-saccharide used to prepare the composition is selected from thegroup consisting of trehalose dihydrate, anhydrous trehalose, sucrose,raffinose and combinations thereof.
 11. The method according to claim 8,wherein the non-reducing disaccharide used to prepare the composition isselected from sucrose, trehalose dihydrate or anhydrous trehalose. 12.The method according to claim 11, wherein the non-ionic surfactant usedto prepare the composition is selected from the group consisting ofpolysorbates, sorbitan esters, octylphenol ethylene oxide nonylphenolethoxylate, poloxamers and combinations thereof.
 13. The methodaccording to claim 12, wherein the non-ionic surfactant used to preparethe composition is selected from the group consisting of polysorbate 20,polysorbate 40, polysorbate 60, polysorbate 80, sorbitan monolaurate,sorbitan monostearate, sorbitan tristearate, and sorbitan monooleate.14. The method according to claim 13, wherein the physiologicallycompatible buffer is selected from the group consisting of citric acid,acetic acid, succinic acid, tartaric acid, maleic acid, histidine,citrate/acetate, citrate/histidine, citrate/tartrate, maleate/histidine,succinate/histidine, or salts thereof, and phosphate buffer.
 15. Themethod according to claim 14, wherein the positively charged backbone isthe peptide with an amino acid sequence selected fromRKKRRQRRR-G-(K)15-G-RKKRRQRRR (SEQ ID NO: 1),RGRDDRRQRRR-G-(K)15-G-RGRDDRRQRRR (SEQ ID NO: 2), orYGRKKRRQRRR-G-(K)15-G-YGRKKRRQRRR (SEQ ID NO: 3).
 16. The methodaccording to claim 14, wherein the positively charged backbone ispolylysine having attached thereto at least one positively chargedefficiency group selected from amino acid sequence(gly)p-RKKRRQRRR-(gly)q (SEQ ID NO: 5); (gly)p-RGRDDRRQRRR-(gly)q (SEQID NO: 6); or (gly)p-YGRKKRRQRRR-(gly)q (SEQ ID NO: 7); wherein thesubscripts p and q are each independently an integer of from 0 to 20.17. The method according to claim 16, wherein the subscript n1 is aninteger of from 2 to 5; and the subscript n2 is independently an oddinteger of from 7 to
 13. 18. The method according to claim 16, whereinthe subscripts p and q are each independently an integer of from 2 to 5.19. The method according to claim 1, wherein the concentration of thenon-reducing disaccharide is in the range of 10% to 40%.